1 Introduction:

In photosynthesis, a blue/green substance called chlorophyll A and a yellow/green substance called chlorophyll B use light energy (normally sunlight but sometimes artificial) to change carbon dioxide and water into sugars (carbohydrates) and oxygen in the green parts of the plant. The amount of photosynthesis per day which takes place is limited by the duration and intensity of sunlight, and the ability of the green parts of a plant to capture it. The amount of carbon dioxide available can also be a limiting factor. Shortage of water, low temperatures, and leaf disease or damage can reduce photosynthesis, as can shading by other plants, e.g. by weeds in a crop. The cells that contain chlorophyll also have orange/yellow pigments such as xanthophyll and carotene, and brown pigments called pheophytins which absorb different wavelengths of light than the chlorophylls. Crop plants can only build up chlorophyll A and B in the light, and so any leaves that develop in the dark are yellow and cannot efficiently produce carbohydrates. The yellowing of leaves (chlorosis) can also be caused by disease attack, nutrient deficiency or natural senescence (dying off).Chloro- phylls a and b (Chl a, Chl b) represent the majority of the antenna complex pigments and thus are of great im- portance for light absorption, oxygen evolution, and conversion of light energy to chemical energy. In fact, the amount of solar radiation that is absorbed by a leaf is closely related to its chlorophyll concentration (Huang et al. 2014; Gitelson et al. 2014; Samet and Sinclair 1980), which generally is positively related with photosynthetic rate.

Generally, plants modulate leaf anatomy and physiology to irradiance, developing thicker leaves with a greater mesophyll to surface-area ratio (Boardman 1977; Lichtenthaler et al. 1981; Anderson 1986). Common adjustments to high irradiance also include a reduction in the total Chl per unit leaf area and an increased Chl a/b ratio (Bjorkman 1972; Boardman 1977; Lichtenthaler et al. 1981; Anderson 1986). Due to its pre- dictable response to irradiance, the Chl a/b ratio has been proposed as a bioassay to assess the light environment of a plant (Dale and Causton 1992). Under crop production conditions, the irradiance of most leaves is influenced by plant den- sity and row distance and modulates various aspects of the photosynthetic apparatus. Thus, genotype specific Chl composition and content characteristics could have implications for crop management.

The main objectives of this project were to assess genotypic variation in soybean Chl composition and to use genome-wide association mapping to identify genomic loci associated with extract-based measurements of chlorophyll a, chlorophyll b, and total chlorophyll content, as well as chlorophyll a/b ratio.

2 Analysis

2.1 Chlorophyll content determinations

Chlorophyll contents were determined using extract- and canopy reflectance-based methods. The chlorophyll contents determined from extracts of leaf disks are here- after referred to as chlorophyll a (Chl A), chlorophyll b (Chl B), chlorophyll a/b ratio (Chl Ratio) and total chlorophyll (Chl Total). Briefly, at 54 days after planting (DAP; 2009) and 60 DAP (2010), five 0.68 cm2 leaf disks were collec- ted from the upper-most fully expanded, sun-exposed leaf (3rd or 4th leaf from the stem apex) from five differ- ent plants at flowering (R1-R2 stage). The leaf disks were immediately placed in opaque glass vials containing 5 mL of ethanol (95 %, v/v). Samples were incubated at room temperature in the dark for 24 h, after which, the vials were vigorously agitated. A 200 \(\mu L\) aliquot of each sample was transferred to a 96 well-plate (Costech Analytical Technologies Inc., CA USA) and absorbance measured at 664, 648, and 470 nm on a Scanning Monochromatic Spectrophotometer (Bio-Tek Power- Wave X 340 Microplate Reader, BioTek U.S. VT, USA). Total chlorophyll (Chl Total), chlorophyll a (Chl A), and chlorophyll b (Chl B) were calculated according to Lichtenthaler, expressed on a leaf-area basis (\(\mu g\) \(cm^2\)). The ratio of Chl A and Chl B was determined and is referred to as Chl Ratio.

Table 2.1: Arconym and References for Chlorophyll
Data analysis Acronym References
Extraction and spectrophotometric measurements
Chlorophyll a ChlA Lichtenthaler 1987
Chlorophyll b ChlB Lichtenthaler 1987
Chlorophyll a/b ratio Chl A Chl B ratio Lichtenthaler 1987
Total chlorophyll ChlAB Total Lichtenthaler 1987

2.2 Method

In this project we applied FARMCPU (Liu et al. 2016) method to find the significant SNPs. FarmCPU is a Genome Wide Association Study (GWAS) method (PLoS Genetics, 2016), standing for “Fixed and random model Circulating Probability Unification”. FarmCPU join the advantages of mixed linear model and stepwise regression (fixed effect model) and overcome their disadvantages by using them iteratively. To eliminate the confounding between kinship in a mixed model (MLM) and genes underlying a trait of interest, FarmCPU substitutes kinship with a set of markers associated with the causal genes. The set of the associated markers are fitted as fixed effect in a fixed effect model for testing markers one at a time across genome. To avoid model overfitting for testing markers, the set of associated markers are optimized in a maximum likelihood method in an MLM with variance and covariance structure defined by the associated markers. Both computer simulation and real data analyses demonstrated that FarmCPU has higher power and less false positives than either MLM or stepwise regression. FARMCPU was run by R package GAPITv3 in our project.

2.3 Data

The genotypic data for the 818 soybean accessions (version Wm82.a2) was obtained from the application of the SoySNP50K iSelect BeadChip (Song et al. 2013). In total, 42195 polymorphic SNPs with MAF \(\ge\) 0.01 accross 818 genotypes were used for GWAS mapping of ChlA, ChlB, ChlAB Total, ChlA ChlB ratio. Soybase GWAS position and gene anotation were obtained from (https://soybase.org). Soybase Gene Model positions Glyma we used in this analysis is version 2.0.

The distribution of Chl A, Chl B, and Total Chl look normal distributed in the Fig. , the distribution of Chl A Chl B ratio is bi-bellshape distributed. As in the Table , Chl A and Chl B and Total Chl are highly correllated, and there is not much relations Chl ratio with others.

Table 2.2: Correllation of measurement of different Chl types
Name Total Chl Chl A Chl B Chl ratio
Total Chl 1.0000000 0.9886897 0.9066737 -0.2910365
Chl A 0.9886897 1.0000000 0.8331543 -0.1491333
Chl B 0.9066737 0.8331543 1.0000000 -0.6537444
Chl ratio -0.2910365 -0.1491333 -0.6537444 1.0000000

2.4 Results

As results, we obtained that at the \(\alpha = 0.05\) level of significant, there are 25 SNPs with MAF \(\ge\) 0.01 were identified having significant associations with ChlA, ChlB, ChlAB Total levels and ChlA ChlB ratio. There are 14 SNPs (Table ) identified in 9 loci (Table ) associated with ChlA ChlB ratio, 4 SNPs (Table ) identified in 4 loci(Table ) associated with ChlA, 7 SNPs (Table ) identified in 3 loci(Table ) associated with ChlB and 2 SNPs (Table ) indentified in 2 loci(Table ) associated with Total ChlA ChlB. There are 2 SNPs associated to both ChlA ChlB ratio and ChlB, the other pairs have no commond association. We also observe that the results do not change if we choose the significant level to 0.01 (Fig. , , ,).

Box plots and histogram for Chls

Figure 2.1: Box plots and histogram for Chls

Table 2.3: Phenotype Variance Explained by Association Markers by Chl types
SNP Chromosome Position P.value maf effect Variance_Explained Type
ss715579621 1 47717883 0e+00 0.1210269 -0.0327613 0.0179808 Chl A/B ratio
ss715580343 1 53141084 0e+00 0.3007335 -0.0807571 0.0338340 Chl A/B ratio
ss715580344 1 53151056 0e+00 0.2866748 -0.0246138 0.0042362 Chl A/B ratio
ss715580552 1 55263448 2e-07 0.0550122 0.0329895 0.0026452 Chl A/B ratio
ss715590313 5 4423839 0e+00 0.3349633 -0.0559207 0.0009964 Chl A/B ratio
ss715590996 5 34335534 0e+00 0.3044010 -0.0299305 0.0150065 Chl A/B ratio
ss715602773 8 8571552 0e+00 0.2542787 0.0201908 0.0040270 Chl A/B ratio
ss715602901 8 9837263 0e+00 0.1674817 0.0661252 0.0022085 Chl A/B ratio
ss715599152 8 10102541 0e+00 0.2310513 0.0016118 0.0022100 Chl A/B ratio
ss715610699 11 4259267 0e+00 0.3783619 0.0181413 0.0168382 Chl A/B ratio
ss715610815 11 5060627 0e+00 0.3734719 0.0254823 0.0024868 Chl A/B ratio
ss715609725 11 13764673 1e-07 0.0226161 -0.1407266 0.0051594 Chl A/B ratio
ss715609736 11 14122451 0e+00 0.0244499 -0.0622868 0.0039476 Chl A/B ratio
ss715610196 11 31286570 0e+00 0.0201711 0.0347399 0.0000000 Chl A/B ratio
ss715581005 2 11686919 0e+00 0.2218826 1.3176296 0.0031495 Chl A
ss715598780 7 86898 0e+00 0.0354523 0.2716099 0.0406689 Chl A
ss715599055 7 9535961 1e-07 0.2701711 1.3153512 0.0000000 Chl A
ss715624377 16 31172215 0e+00 0.0152812 -4.7230635 0.1614091 Chl A
ss715580343 1 53141084 0e+00 0.3007335 -0.1594132 0.0000000 Chl B
ss715580344 1 53151056 0e+00 0.2866748 -0.6818936 0.0147080 Chl B
ss715598601 7 7349078 0e+00 0.3306846 -0.3663648 0.0000000 Chl B
ss715598602 7 7354616 0e+00 0.3312958 0.3977816 0.0095525 Chl B
ss715597527 7 37049207 1e-07 0.4425428 0.0951097 0.0083861 Chl B
ss715623433 16 13285061 1e-07 0.1185819 -0.5628720 0.0142384 Chl B
ss715627201 17 37093778 0e+00 0.1393643 0.5833435 0.0214669 Chl B
ss715581877 2 29620600 1e-07 0.3539120 1.5139055 0.0029779 Total Chl A Chl B
ss715604090 9 3931954 1e-07 0.0525672 2.9529925 0.1118926 Total Chl A Chl B

The Vienn diagram Fig. shows us the relations of significant SNPs among Chl A, Chl B, Chl AB total and Chl A/B ratio.

Venn diagram for relations of Chl types

Figure 2.2: Venn diagram for relations of Chl types

There are (14, 4, 7, 2 ) SNPs that are significant associated (respectively) to ChlA ChlB ratio, ChlA, ChlB and Total Chl. However, there is no significant SNPs that belong to all 4 catergories.

According to Soybase GWAS position dataset obtained from website (https://soybase.org), our results show that there is only one SNP (table ) that is significant explained by the Chlorophyll ratio and there is no SNP that is in Soybase GWAS position dataset associated with Chl A, Total Chl, and Chl B. The following table is the detail of the significant SNPs.

Table 2.4: Significant SNPs identified in GWAS position dataset
SNP Chromosome Position P.value maf effect Variance_Explained Trait
ss715590996 5 34335534 0 0.304401 -0.0299305 0.0150065 Chl A Chl B ratio
Table 2.5: List of soybase CHL genes and their position that is in 3 MB from SNP in ChlA ChlB ratio Phenotype Variance Explained by Association Marker
Loci Feature Name Start End Distance to SNP Functional annotation
1 Glyma.01g153500 49096486 49097279 1.378603 Photosynthetic reaction centre protein
2 Glyma.01g204700 53740048 53741473 0.598964 Chlorophyll A-B binding protein
Glyma.01g226700 55505550 55515233 2.364466 von Willebrand factor type A domain; Magnesium chelatase, subunit ChlI
3 Glyma.05g026200 2281768 2284008 2.139831 Pyridine nucleotide-disulphide oxidoreductase
Glyma.05g066100 6479570 6481579 2.055731 sequence-specific DNA binding transcription factor activity
4 Glyma.05g128000 32149719 32150849 2.184685 Chlorophyll A-B binding protein
Glyma.05g129700 32281938 32287791 2.047743 MCM2/3/5 family
Glyma.05g149400 34365927 34369714 0.030393 PRONE (Plant-specific Rop nucleotide exchanger); Chlorophyll A-B binding protein
Glyma.05g156300 34881112 34887058 0.545578 Pheophorbide a oxygenase; Rieske [2Fe-2S] domain
5 Glyma.08g074000 5651670 5653067 2.918485 Chlorophyll A-B binding protein
Glyma.08g082900 6276579 6277935 2.293617 Chlorophyll A-B binding protein
Glyma.08g084400 6360443 6366566 2.204986 MCM2/3/5 family
Glyma.08g114300 8746643 8747874 0.175091 IRON-SULFUR DOMAIN CONTAINING PROTEIN
Glyma.08g114400 8750828 8756629 0.179276 Pheophorbide a oxygenase; Rieske [2Fe-2S] domain
7 Glyma.11g101800 7719377 7722788 2.658750 GTP-BINDING PROTEIN-RELATED
8 Glyma.11g149600 11642938 11645593 2.119080 molecular function
9 Glyma.11g228800 32398995 32402927 1.112425 Chlorophyll A-B binding protein

We found that the following genes Glyma01g41320, Glyma01g43630, Glyma05g01000, Glyma05g05450 in our results were also identified by (Dhanapal et al. 2016).

Among the significant SNPs, there are 4 SNPs ss715580343, ss715580344, ss715598602, ss715604090in exon regions.

Table 2.6: List of soybase CHL genes that is within 3MB from ChlA Phenotype Variance Explained by Association Marker SNP
Loci Feature Name Start End Distance to SNP Functional annotation
1 Glyma.02g134200 13867709 13869124 2.180790 alpha/beta hydrolase fold
2 Glyma.07g000500 62458 75399 0.011499 NmrA-like family; Complex I intermediate-associated protein 30 (CIA30)
Glyma.07g029900 2383023 2386348 2.296125 molecular function
3 Glyma.07g085700 7910707 7915213 1.620748 short chain dehydrogenase
Glyma.07g096800 9079793 9081431 0.454530 Chlorophyllase
Glyma.07g096900 9083837 9085839 0.450122 Chlorophyllase
Glyma.07g097000 9118318 9120182 0.415779 Chlorophyllase
4 Glyma.16g132500 28902729 28905667 2.266548 alpha/beta hydrolase fold
Glyma.16g138000 29508469 29514252 1.657963 alpha/beta hydrolase fold
Glyma.16g145800 30677170 30679559 0.492656 Chlorophyll A-B binding protein
Glyma.16g162600 32154717 32155227 0.982502 Chlorophyll A-B binding protein
Glyma.16g165200 32442602 32444100 1.270387 Chlorophyll A-B binding protein
Glyma.16g165500 32457671 32458673 1.285456 Chlorophyll A-B binding protein
Glyma.16g165800 32484354 32486237 1.312139 Chlorophyll A-B binding protein
Table 2.7: List of soybase CHL genes that is within 3MB from ChlB Phenotype Variance Explained by Association Marker SNP
Loci Feature Name Start End Distance to SNP Functional annotation
1 Glyma.01g204700 53740048 53741473 0.598964 Chlorophyll A-B binding protein
Glyma.01g226700 55505550 55515233 2.364466 von Willebrand factor type A domain; Magnesium chelatase, subunit ChlI
2 Glyma.07g085700 7910707 7915213 0.561629 short chain dehydrogenase
Glyma.07g096800 9079793 9081431 1.730715 Chlorophyllase
Glyma.07g096900 9083837 9085839 1.734759 Chlorophyllase
Glyma.07g097000 9118318 9120182 1.769240 Chlorophyllase
3 Glyma.07g204300 37333134 37335896 0.283927 Magnesium chelatase, subunit ChlI
Glyma.07g211000 38220165 38221079 1.170958 alpha/beta hydrolase fold
Table 2.8: List of soybase CHL genes that is within 3MB from Total ChlA ChlB Phenotype Variance Explained by Association Marker SNP
Loci Feature Name Start End Distance to SNP Functional annotation
1 Glyma.02g170600 26652157 26657142 2.963458 MCM2/3/5 family
2 Glyma.09g047300 4077473 4083792 0.145519 MCM2/3/5 family

Table 2.9: Identification and information of SNPs significantly associated to all type of Chl Phenotype Association Markers with maf > 0.01
SNP Chr P-value effect Var. Explained MAF Minor Adj.FDR Type
BARC_1.01_Gm01_46841836_T_C 1 0e+00 -0.0327613 0.0179808 0.1210269 C 0.0000187 Chl A/B ratio
BARC_1.01_Gm01_46841836_T_C 1 0e+00 -0.0327613 0.0179808 0.1210269 T 0.0000187 Chl A/B ratio
BARC_1.01_Gm01_52253980_C_T 1 0e+00 -0.0807571 0.0338340 0.3007335 T 0.0000000 Chl A/B ratio
BARC_1.01_Gm01_52253980_C_T 1 0e+00 -0.0807571 0.0338340 0.3007335 C 0.0000000 Chl A/B ratio
BARC_1.01_Gm01_52263952_T_C 1 0e+00 -0.0246138 0.0042362 0.2866748 C 0.0000000 Chl A/B ratio
BARC_1.01_Gm01_52263952_T_C 1 0e+00 -0.0246138 0.0042362 0.2866748 T 0.0000000 Chl A/B ratio
BARC_1.01_Gm01_54354945_G_A 1 2e-07 0.0329895 0.0026452 0.0550122 G 0.0005466 Chl A/B ratio
BARC_1.01_Gm01_54354945_G_A 1 2e-07 0.0329895 0.0026452 0.0550122 A 0.0005466 Chl A/B ratio
BARC_1.01_Gm05_2708074_T_C 5 0e+00 -0.0559207 0.0009964 0.3349633 T 0.0000079 Chl A/B ratio
BARC_1.01_Gm05_34064933_C_T 5 0e+00 -0.0299305 0.0150065 0.3044010 T 0.0000079 Chl A/B ratio
BARC_1.01_Gm08_8541523_G_T 8 0e+00 0.0201908 0.0040270 0.2542787 T 0.0000337 Chl A/B ratio
BARC_1.01_Gm08_8541523_G_T 8 0e+00 0.0201908 0.0040270 0.2542787 G 0.0000337 Chl A/B ratio
BARC_1.01_Gm08_9845186_T_C 8 0e+00 0.0661252 0.0022085 0.1674817 C 0.0000275 Chl A/B ratio
BARC_1.01_Gm08_9845186_T_C 8 0e+00 0.0661252 0.0022085 0.1674817 T 0.0000275 Chl A/B ratio
BARC_1.01_Gm08_10189778_C_T 8 0e+00 0.0016118 0.0022100 0.2310513 T 0.0001441 Chl A/B ratio
BARC_1.01_Gm08_10189778_C_T 8 0e+00 0.0016118 0.0022100 0.2310513 C 0.0001441 Chl A/B ratio
BARC_1.01_Gm11_4249856_G_A 11 0e+00 0.0181413 0.0168382 0.3783619 G 0.0000000 Chl A/B ratio
BARC_1.01_Gm11_4249856_G_A 11 0e+00 0.0181413 0.0168382 0.3783619 A 0.0000000 Chl A/B ratio
BARC_1.01_Gm11_5051077_T_G 11 0e+00 0.0254823 0.0024868 0.3734719 G 0.0000253 Chl A/B ratio
BARC_1.01_Gm11_5051077_T_G 11 0e+00 0.0254823 0.0024868 0.3734719 T 0.0000253 Chl A/B ratio
BARC_1.01_Gm11_24232656_C_T 11 1e-07 -0.1407266 0.0051594 0.0226161 T 0.0002199 Chl A/B ratio
BARC_1.01_Gm11_24590514_T_C 11 0e+00 -0.0622868 0.0039476 0.0244499 C 0.0001444 Chl A/B ratio
BARC_1.01_Gm11_24590514_T_C 11 0e+00 -0.0622868 0.0039476 0.0244499 T 0.0001444 Chl A/B ratio
BARC_1.01_Gm11_35762107_A_G 11 0e+00 0.0347399 0.0000000 0.0201711 G 0.0001444 Chl A/B ratio
BARC_1.01_Gm11_35762107_A_G 11 0e+00 0.0347399 0.0000000 0.0201711 A 0.0001444 Chl A/B ratio
BARC_1.01_Gm02_32796990_G_A 2 1e-07 1.5139055 0.0029779 0.3539120 A 0.0011014 Total Chl
BARC_1.01_Gm09_3889437_T_C 9 1e-07 2.9529925 0.1118926 0.0525672 C 0.0011014 Total Chl
BARC_1.01_Gm01_52253980_C_T 1 0e+00 -0.1594132 0.0000000 0.3007335 T 0.0000805 Chl B
BARC_1.01_Gm01_52253980_C_T 1 0e+00 -0.1594132 0.0000000 0.3007335 C 0.0000805 Chl B
BARC_1.01_Gm01_52263952_T_C 1 0e+00 -0.6818936 0.0147080 0.2866748 C 0.0000000 Chl B
BARC_1.01_Gm01_52263952_T_C 1 0e+00 -0.6818936 0.0147080 0.2866748 T 0.0000000 Chl B
BARC_1.01_Gm07_7309830_T_C 7 0e+00 -0.3663648 0.0000000 0.3306846 T 0.0000001 Chl B
BARC_1.01_Gm07_7309830_T_C 7 0e+00 -0.3663648 0.0000000 0.3306846 C 0.0000001 Chl B
BARC_1.01_Gm07_7315368_T_C 7 0e+00 0.3977816 0.0095525 0.3312958 T 0.0001274 Chl B
BARC_1.01_Gm07_7315368_T_C 7 0e+00 0.3977816 0.0095525 0.3312958 C 0.0001274 Chl B
BARC_1.01_Gm07_37143613_T_C 7 1e-07 0.0951097 0.0083861 0.4425428 C 0.0005649 Chl B
BARC_1.01_Gm16_13425509_T_C 16 1e-07 -0.5628720 0.0142384 0.1185819 T 0.0003426 Chl B
BARC_1.01_Gm16_13425509_T_C 16 1e-07 -0.5628720 0.0142384 0.1185819 C 0.0003426 Chl B
BARC_1.01_Gm17_37382483_A_G 17 0e+00 0.5833435 0.0214669 0.1393643 G 0.0000652 Chl B
BARC_1.01_Gm02_11350199_T_G 2 0e+00 1.3176296 0.0031495 0.2218826 G 0.0005785 Chl A
BARC_1.01_Gm07_9498944_A_G 7 1e-07 1.3153512 0.0000000 0.2701711 A 0.0012218 Chl A
BARC_1.01_Gm07_9498944_A_G 7 1e-07 1.3153512 0.0000000 0.2701711 G 0.0012218 Chl A

3 SNPs distribution and Manhattan plot of significant SNPs by FarmCPU algorithm

Distribution of SNPs in Chromosomes

Figure 3.1: Distribution of SNPs in Chromosomes

Significant SNPs and their Chl types with their positions in Chromosomes

Figure 3.2: Significant SNPs and their Chl types with their positions in Chromosomes

Manhattan plot for Chl ratio significants

Figure 3.3: Manhattan plot for Chl ratio significants

Manhattan plot for Chl A significants

Figure 3.4: Manhattan plot for Chl A significants

Manhattan plot for Chl B significants

Figure 3.5: Manhattan plot for Chl B significants

Manhattan plot for Total Chl significants

Figure 3.6: Manhattan plot for Total Chl significants

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